Role of the origin of transfer in termination of strand transfer during bacterial conjugation

Abstract

Conjugal transfer of the broad-host-range plasmid R1162 is initiated and terminated at the nic site within the 38-bp origin of transfer (oriT). Termination involves ligation of the transferred single strand by the plasmid-encoded MobA protein. Several different assays were used to identify the oriT DNA required for termination. For plasmids containing two oniTs, with transfer initiated at one and terminated at the other, the inverted repeat within oniT is-important for termination. Deletion of the outer arm reduces the termination frequency-, those terminations that do occur probably depend upon nicking at this oriT prior to transfer. The locations of second-site suppressor mutations indicate that base pairing between the arms of the inverted repeat is important for termination. In vitro, the inverted repeat is not required for specific cleavage of single-stranded DNA at nic, but competition experiments indicate that oriTs with the inverted repeat are preferentially cleaved.We propose that the function of the oriT inverted repeat is to trap the plasmid-encoded MobA protein at the end of a round of strand transfer, thus ensuring that the protein is available for the ligation step.The basic steps in DNA processing during conjugal transfer are probably similar for many different plasmids in gram-negative bacteria. One of the DNA strands is first nicked within the origin of transfer (oriT), which is required in cis. The nicked strand is then linearly transferred into a recipient cell in the 5'-to-3' direction and subsequently recircularized. In both donor and recipient cells, the complementary strand of DNA is resynthesized to restore the duplex plasmid molecule. Many different plasmids can be isolated in the form of a relaxosome, a complex of proteins and supercoiled plasmid DNA, which can be induced to undergo site-and strand-specific nicking within oriT (for example, see references 5, 6, 12, and 16). This nicking models the initial DNA-processing step in plasmid transfer.R1162 (nearly identical to RSF1010) is a broad-host-range, IncQ plasmid that is efficiently mobilized during the conjugal transfer of IncPl group plasmids (15,20). The R1162 oriT consists of no more than 38 bp of DNA (Fig.

Conjugative transfer in the dissemination of beta-lactam and aminoglycoside resistance

“…A different termination nicking reaction for R1162 was engineered by placing a second oriT "downstream" from the first, in terms of the direction of DNA transfer. The termination/recircularization event at the second oriT was prevented if the inverted repeat within this oriT was removed, consistent with the hypothesis described above (81).…”

The relaxosome protein MobC promotes conjugal plasmid mobilization by extending DNA strand separation to the nick site at the origin of transfer

“…We have suggested that these steps involve the same reaction, carried out by the MobA protein and requiring a similar substrate (Zhang and Meyer, 1995). In each case, MobA cleaves within a single-stranded region, but requires adjacent duplex DNA at the inner arm of the inverted repeat for tight binding (Bhattacharjee et al, 1992;Bhattacharjee and Meyer, 1993). In the case of the single, transferred strand, a duplex structure adjacent to the cleavage site is generated by basepairing between the arms of the inverted repeat (Barlett et al, 1990;Bhattacharjee and Meyer, 1993).…”

“…Plasmids used in this study are listed in Table 3. Conjugal matings were carried out on semi-solid media essentially as described in previous publications (Brasch and Meyer, 1986;Bhattacharjee et al, 1992), with donor MV10 or LE316 cells containing the IncP-1 plasmid R751 as mobilizing vector (Willetts and Crowther, 1981). The recipient strain was DF1019, a C600 derivative resistant to nalidixic acid (Figurski et al, 1976).…”

Initiation and Termination of DNA Transfer during Conjugation of IncI1 Plasmid R64: Roles of Two Sets of Inverted Repeat Sequences within oriT in Termination of R64 Transfer

“…Three R1162 proteins, MobA, MobB, and MobC, form a protein-DNA complex called the relaxosome at oriT (27). From the mobilization experiments with recombinant plasmids containing two tandemly repeated oriT sequences with or without mutations, the 10-bp inverted repeat structure as well as the sequence around the nick site was shown to be required for termination, while the nick site-distal arm of the inverted repeat was not required for initiation (1). From the analyses of mutations introduced into the 10-bp repeat, it has been postulated that during termination of DNA transfer, formation of a hairpin loop structure by the inverted repeat is required for the resealing of the transferred DNA by the R1162 MobA relaxase (32).…”